The focus of this proposal is to study the effect of higher ordered actin structure and of actin binding proteins for the processivity and movement behavior of myosin V molecule. Recently, many in vitro motility studies investigated how myosin V moves on actin filament, measuring parameters such as the run length, velocity and step size, but these studies almost always have used only single actin filaments for the track. However, actin filaments form many kinds of higher ordered structures such as branching networks, loose bundles and highly ordered bundles inside cells. This study will give insight into fundamental questions such as can myosin V step laterally from one filament to another? when moving on a bundle? Does this result in longer run lengths than observed on single filaments? Does binding of tropomyosin to actin affect the velocity or run length? To answer these questions, I will use three different actin formations. As AIM 1, (1) single actin filaments decorated by nonmuscle tropomyosin, or the actin binding proteins, fascin, and a-actinin. (2) Two dimensional paracrystalline arrays of actin filaments bundled on lipid monolayers with a-actinin or fascin. (3) Three dimensional bundles of actin filaments mediated by a-actinin or fascin formed in solution. I will measured run-length and speed of myosin V on two dimentional actin bundles and will use to test for a fiexibility of the myosin neck using these above different types of actin tracks. As AIM 2,1 will prepare Triton-insoluble cytoskeletons by treatment of cells growing on a coverslip surface by treatment with Triton X-100 to remove membranes and with rhodamine phalloidin to stabilize the acfin filaments. The movement of fiuorescently labeled myosin V molecules will be measured using total internal reflection fiuorescent microscopy and an analysis technique, termed FIONA (Fluorescent Imaging at One Nanometer Accuracy) which measures the position of a single fiuorophor to 1.5 nm accuracy with 0.5 s temporal resolution. In AIM 1 and 2,1 will answer how does myosin V step over different kinds of actin bundles and how does myosin V move along a highly ordered actin structure as a transporter. AIM 3,1 have completed thi project.